Preparation of Peptide and Recombinant Tissue Plasminogen Activator Conjugated Poly(Lactic-Co-Glycolic Acid)

International Journal of Molecular Sciences Article Preparation of Peptide and Recombinant Tissue Plasminogen Activator Conjugated Poly(Lactic-Co-Glycolic Acid) (PLGA) Magnetic Nanoparticles for Dual Targeted Thrombolytic Therapy Huai-An Chen 1, Yunn-Hwa Ma 2 , Tzu-Yuan Hsu 2 and Jyh-Ping Chen 1,3,4,5,* 1 Department of Chemical and Materials and Materials Engineering, Chang Gung University, Kwei-San, Taoyuan 33302, Taiwan; [email protected] 2 Department of Physiology and Pharmacology and Healthy Aging Research Center, Chang Gung University, Kwei-San, Taoyuan 33302, Taiwan; [email protected] (Y.-H.M.); [email protected] (T.-Y.H.) 3 Department of Plastic and Reconstructive Surgery and Craniofacial Research Center, Chang Gung Memorial Hospital, Linkou, Kwei-San, Taoyuan 33305, Taiwan 4 Research Center for Food and Cosmetic Safety, Research Center for Chinese Herbal Medicine, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 33302, Taiwan 5 Department of Materials Engineering, Ming Chi University of Technology, Tai-Shan, New Taipei City 24301, Taiwan * Correspondence: [email protected]; Tel.: +886-3211-8800 (ext. 5298) Received: 10 March 2020; Accepted: 10 April 2020; Published: 13 April 2020 Abstract: Recombinant tissue plasminogen activator (rtPA) is the only thrombolytic agent that has been approved by the FDA for treatment of ischemic stroke. However, a high dose intravenous infusion is required to maintain effective drug concentration, owing to the short half-life of the thrombolytic drug, whereas a momentous limitation is the risk of bleeding. We envision a dual targeted strategy for rtPA delivery will be feasible to minimize the required dose of rtPA for treatment. For this purpose, rtPA and fibrin-avid peptide were co-immobilized to poly(lactic-co-glycolic acid) (PLGA) magnetic nanoparticles (PMNP) to prepare peptide/rtPA conjugated PMNPs (pPMNP-rtPA). During preparation, PMNP was first surface modified with avidin, which could interact with biotin. This is followed by binding PMNP-avidin with biotin-PEG-rtPA (or biotin-PEG-peptide), which was prepared beforehand by binding rtPA (or peptide) to biotin-PEG-maleimide while using click chemistry between maleimide and the single –SH group in rtPA (or peptide). The physicochemical property characterization indicated the successful preparation of the magnetic nanoparticles with full retention of rtPA fibrinolysis activity, while biological response studies underlined the high biocompatibility of all magnetic nanoparticles from cytotoxicity and hemolysis assays in vitro. The magnetic guidance and fibrin binding effects were also confirmed, which led to a higher thrombolysis rate in vitro using PMNP-rtPA or pPMNP-rtPA when compared to free rtPA after static or dynamic incubation with blood clots. Using pressure-dependent clot lysis model in a flow system, dual targeted pPMNP-rtPA could reduce the clot lysis time for reperfusion by 40% when compared to free rtPA at the same drug dosage. From in vivo targeted thrombolysis in a rat embolic model, pPMNP-rtPA was used at 20% of free rtPA dosage to restore the iliac blood flow in vascular thrombus that was created by injecting a blood clot to the hind limb area. Keywords: magnetic nanoparticles; tissue plasminogen activator; poly(lactic-co-glycolic acid); targeted drug delivery; clot lysis; nanomedicine Int. J. Mol. Sci. 2020, 21, 2690; doi:10.3390/ijms21082690 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 2690 2 of 23 1. Introduction Within the two ischemic and hemorrhagic categories of stroke, the ischemic stroke accounts for ~87% of all cases, according to the American Stroke Association [1]. A multitude of molecular pathways may be involved in the onset and progression of ischemic stroke, thus an equally diverse arsenal of intervention strategies is needed. To date, the gold standard for intravenous (IV) intervention of ischemic stroke is by administrating recombinant tissue plasminogen activator (rtPA), a thrombolytic drug that dissolves clots to restore blood flow. As a serine protease, rtPA is the major enzyme that is responsible for clot dissolution by catalyzing the conversion of plasminogen to plasmin. It is currently the only thrombolytic agent approved by the U.S. Food and Drug Administration (FDA) for the treatment of ischemic stroke [2]. A major issue in rtPA therapeutic use is the short half-life (4–8 min) in the circulation due to autolysis and the influences of inhibitors, enzymes, and antibodies in blood [3]. The poor affinity of rtPA toward thrombus also reduces its thrombolysis efficacy and might cause ischemia reperfusion injury to the neuron beyond the thrombolysis window [4]. Other limitations of rtPA for clinical use include the short window time for treatment (usually within 3 h) and the risk of hemorrhagic side effects [5]. Consequently, several strategies have emerged to improve the thrombolytic efficacy of rtPA, such as ultrasound-based thrombolysis, targeting thrombolysis, and immobilized thrombolytic drug to improve the safety and effectiveness of thrombolytic therapy [6]. Nanomedicine using nanoparticles for drug delivery is a novel field for the diagnosis and treatment of diseases. Although with similar biologic molecular scale when compared with traditional medicine, the unique properties of nanoparticles provide more strategic advantages and application flexibility over pure molecular therapeutics [7]. Using nanoparticles as rtPA carrier for thrombolytic therapy has also been widely explored. For example, biodegradable polymers conjugated with rtPA can provide the protection form the inhibitor in the circulation to prolong its circulation time, which might also confer the possibility for controlled release in encapsulated drug formulation [8]. In recent years, magnetic nanoparticles (MNP), especially iron oxide (Fe3O4) MNP, are frequently employed as a nano-carrier for drug delivery. Not only to be useful as a magnetic resonance imaging contrast agent, the MNP can also be endowed with other advantages, such as magnetic targeting (physical targeting) and localized heating by magnetic field induction or near-infrared laser irradiation [9]. In addition to pristine nanoparticles, MNP could be also surface modified to enhance their functionality as a drug delivery vehicle. The most common example is surface modification with polyethylene glycol (PEG) to prevent uptake by the mononuclear phagocyte system during circulation, thus prolonging the life time of injected drug [10]. Conjugation with ligand is another commonly used strategy for nanoparticle-based targeted drug delivery. Indeed, ligand-mediated drug delivery could offer a highly specific binding between the ligand molecule immobilized to the nano-carrier and the receptor molecule highly expressed in the diseased area, which could enhance the treatment efficacy by inducing drug accumulation and increasing local drug concentration in the targeted area [11]. Many groups have proposed nanomedicine using targeted rtPA delivery strategy for thrombolysis [12]. We have extensively studied different MNP-based polymeric nano-carrier for magnetically targeted delivery of rtPA, being prepared either by immobilization of rtPA on the particle surface or by encapsulation within the polymeric matrix, and demonstrated the improved thrombolytic efficacy both in vitro and in vivo [13–15]. Recently, we also pioneered the use of thermosensitive magnetic liposomes for targeted delivery and temperature-sensitive release of rtPA [16,17]. Other groups used different ligands for targeting fibrin in a blood clot or different moieties that are associated with the blood clot. For example, the ligands used for rtPA delivery targeting thrombus include peptide targeting FXIII [18], fibrin antibody targeting fibrin [19], Arg-Gly-Asp (RGD) [20], cyclicRGD [21], or other peptides [22] targeting GP IIb/IIIa, and fucoidan targeting p-selectin [23]. Those targeted thrombolysis strategies have demonstrated promising results in improving thrombolytic efficiency with shorter clot lysis time, which makes possible the use of reduced drug dosage to circumvent associated side effects, such as the bleeding risk [24]. Int. J. Mol. Sci. 2020, 21, 2690 3 of 23 Consider a ligand targeting fibrin, which is a trimeric molecule consisting of α-, β-, and γ-chains and a major constituent of fresh or old blood clots. Although the actual quantity of fibrin content in a clot varies from clot to clot, fibrin is the major component in a clot and it exists on the surface of a clot that is slowly dissolving either spontaneously or during therapeutic thrombolytic intervention. It is conceivable that a peptide specific for fibrin could be labelled with radioisotopes and used as an imaging agent to detect vascular thrombosis. This was demonstrated from a previous study using 99mTc labelled pentapeptide Gly-Pro-Arg-Pro-Pro (GPRPP), which has high affinity for the fibrin α-chain, for the imaging of vascular thrombosis in animals [25]. The peptide GGSKGC was later added to the C-terminus of GPRPP in order to impart functionality on the peptide. This peptide (GPRPPGGSKGC) was later shown to have high fibrin-avid affinity and high resistance to proteolysis [26]. Although using a magnetic field to magnetically guide the movement of MNP for rtPA delivery is promising, the optimization of a magnetic field might be the limiting factor, especially for clinical application. On the other hand, ligand targeting for rtPA delivery might pose challenges, such as specificity and possible immunogenicity of the targeting moiety.

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